How pooled baby rabbit complement supported reproducible GBS vaccine assays

Supporting reproducible vaccine research with consistent complement performance 

The successful development of a vaccine depends not only on inducing a protective immune response but also on the availability of robust, standardised assays capable of accurately measuring antibody function.  

This article analyses the 2023 paper published in Vaccines by researchers at the UK Health Security Agency (UKHSA), which describes how pooling batches of baby rabbit complement helped improve the reproducibility of a standardised opsonophagocytic killing assay (OPKA). 

UKHSA created a pooled reagent from several lots of baby rabbit complement supplied by VH Bio.  Although it was just one element of a much larger optimisation study, the use of pooled complement proved to be an important step towards creating a robust assay that could be reproduced internationally. 

Measuring antibody function 

Group B Streptococcus (GBS) remains one of the leading causes of serious bacterial infection in newborns, making maternal vaccination an important area of research. As vaccine candidates progress through development, researchers need assays that measure not only how much antibody is produced, but whether those antibodies can effectively kill the bacteria. 

The opsonophagocytic killing assay (OPKA) is widely used for this purpose. It measures the ability of vaccine-induced antibodies to promote bacterial killing in the presence of immune cells and complement, making the quality and consistency of the complement reagent an important factor in overall assay performance. 

Tackling batch-to-batch variation 

During development of the standardised OPKA, the UKHSA team evaluated a number of variables, including incubation time, incubation temperature, complement concentration and the use of individual versus pooled batches of baby rabbit complement. 

While most assay conditions had little effect on the final results, they found that individual complement batches introduced measurable variation. To reduce this, the researchers combined multiple batches of baby rabbit complement into a single pooled reagent for use throughout the study. 

As reported in the publication: 

“Use of a pooled batch of complement eliminated the variation observed with individual batches.” 

That pooled complement was then incorporated into the final assay protocol. 

A robust assay that could be shared globally 

The optimised assay used representative GBS clinical isolates, differentiated HL-60 cells and 12.5% pooled baby rabbit complement, alongside a straightforward colony-counting endpoint. Importantly, it was designed to be practical as well as reproducible, avoiding specialist equipment that might limit adoption in other laboratories. 

To assess how transferable the method was, four laboratories across three continents independently performed the assay using the same pooled complement supplied by UKHSA while generating their own bacterial cultures and maintaining their own HL-60 cell lines. 

Despite differences in laboratory equipment and routine procedures, the assay produced strong agreement between laboratories, with Pearson correlation coefficients ranging from 0.88 to 0.94 across the five GBS serotypes tested. Those findings demonstrated that the standardised protocol could deliver consistent results in different research settings. 

Why pooling complement matters 

Biological reagents naturally vary from batch to batch, and complement is no exception. In this study, pooling several complement lots removed the variation observed when individual lots were assessed. Using the same qualified pool throughout the interlaboratory study also ensured that participating laboratories were working with a consistent complement reagent. 

For researchers carrying out bactericidal or opsonophagocytic assays, this can offer several practical benefits, including: 

• Reduced batch-to-batch variation 

• More consistent assay performance 

• Improved confidence in long-term studies 

• Better reproducibility between laboratories 

• More reliable comparison of vaccine candidates 

From research assay to global vaccine development 

The work carried out by UKHSA has gone well beyond the original publication. 

Through the Vaccine Development and Evaluation Centre (VDEC) at Porton Down, the team has continued to build on the assay developed as part of the international GASTON consortium. Today, the OPKA is recognised as a “Gold Standard” method for evaluating functional antibody responses against GBS. UKHSA has also selected and made standard GBS test strains available internationally, helping researchers generate comparable data across public health, academic and industry laboratories. 

The programme continues to evolve. Funding from the Bill & Melinda Gates Foundation is supporting development of a higher-throughput version of the assay suitable for large Phase II and III clinical trials, alongside international reference serum panels with defined opsonophagocytic activity. The assay has also been expanded to include additional GBS serotypes, supporting evaluation of next-generation multivalent vaccines. 

While the VDEC case study does not specifically discuss the complement reagent, it highlights the broader impact of the standardised OPKA. The earlier Vaccines publication showed that pooling baby rabbit complement removed the variability seen between individual complement batches, helping establish the reproducibility needed for an assay that is now supporting vaccine development on an international scale. 

Supporting reproducible vaccine research 

The UKHSA study highlights how careful optimisation of every component in a functional assay can contribute to reliable, reproducible results. In this case, pooling batches of baby rabbit complement helped eliminate a source of variability, supporting the development of a standardised assay that is now being used to advance GBS vaccine research worldwide. 

If you’re developing or refining bactericidal or opsonophagocytic assays, VH Bio can provide Cedarlane’s baby rabbit complement or technical support to help you select the right reagent for your application. 

Explore our range of complement reagents or contact our team by filling in the form below to discuss your assay requirements. 

References  

Leung, S., et al. (2023). Development of A Standardized Opsonophagocytosis Killing Assay for Group B Streptococcus and Assessment in an Interlaboratory Study. Vaccines, 11(11), 1703. https://doi.org/10.3390/vaccines11111703 

UKHSA. (2024, May 24). VDEC is supporting a GBS vaccine to prevent newborn deaths – case study – gov.uk. https://www.gov.uk/government/case-studies/vdec-is-supporting-a-gbs-vaccine-to-prevent-newborn-deaths